The main focus of the proposed research are the mechanisms responsible for maintenance of oxidative phosphorylation (OXPHOS) and protection from oxidative damage. We have hypothesized that the mitochondrial protein frataxin represents a critical mechanism to handle redox active iron safely within the mitochondrion. Lack of yeast frataxin has been shown to result in mitochondrial iron accumulation, loss of mitochondrial DNA integrity, and a global deficiency in the maintenance of iron-sulfur clusters. In humans, frataxin defects lead to a neurodegenerative disease, Friedreich ataxia. These findings indicate that frataxin is involved in iron homeostasis and that loss of this function results in oxidative damage leading to neuronal cell death. Preliminary results indicate that human frataxin can assemble with itself and bind iron in stable and bioavailable form. The immediate goal of this proposal is to elucidate the mechanism of action of human frataxin. The following specific aims are proposed: (1) To carry out site directed mutagenesis of conserved amino acid residues predicted to be critical for the assembly, iron binding, and anti-oxidant properties of human frataxin; (2) To express mutant proteins in E. coli and characterize them by biophysical and biochemical methods; (3) To test the effects of severe mutants on OXPHOS maintenance and sensitivity to oxidative stress in the eukaryotic model, S. cerevisiae. This research should provide new information relevant to the pathogenesis of Friedreich ataxia and the role of oxidative damage in neurodegenerative disorders.